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PubMed2001

"Second-look" cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis of CDAD.

Clostridium difficile is one of the most frequent causes of hospital-acquired diarrhoea. Our objective was to prove that some stool samples with a direct negative cytotoxicity assay may indeed harbour toxigenic C. difficile and that this can be demonstrated by performing a "second-look" cytotoxicity assay using the isolated C. difficile strains. Over an eight-year period (1992-1999), the 8241 stool samples submitted for direct cell culture from patients with suspected C. difficile-associated diarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructose agar. C. difficile strains isolated from samples with a negative direct cell culture assay were re-tested for toxin production "second-look" cell culture assay). Using both methods 6423 samples (78%) were negative. Of the remaining 1818 samples, 127 (7%) yielded C. difficile isolates which were confirmed as non-producers of toxin by both methods, 1437 (85%) were positive in direct cell culture assay, and 254 were positive only after the "second-look" cell culture assay. Thus, our approach allowed us to detect an extra 15% of toxin-producing strains that could have gone undetected otherwise.The combination of direct-cell culture assay, culture for toxigenic C. difficile and "second-look" cell culture assay enhances the potential for diagnosis of CDAD and enables us to be more efficient with our patient care resources.

Bacterial Toxins↗
PubMed2004

Staphylococcus aureus and Clostridium difficile cause distinct pseudomembranous intestinal diseases.

We report simultaneous infections with Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) in a patient with discrete colonic pseudomembranes typical of C. difficile infection, as well as confluent, loosely adherent pseudomembranes in the small bowel. Identification of MRSA in the small bowel pseudomembrane by polymerase chain reaction supports S. aureus as an enteric pathogen.

Clostridioides difficile↗
PubMed1996

Prospective study of toxigenic Clostridium difficile in children given amoxicillin/clavulanate for otitis media.

OBJECTIVE: Evaluate antibiotic-associated diarrhea and toxigenic Clostridium difficile in stool specimens obtained from children before and after 10 days of amoxicillin/clavulanate for otitis media. DESIGN: Children, 12 to 47 months of age, treated with amoxicillin/clavulanate for otitis media in an outpatient setting were enrolled. Stool specimens were obtained at enrollment, when diarrhea occurred and at the end of therapy. All stool specimens were tested for C. difficile toxins A and B by enzyme immunoassay. RESULTS: Seventy-six children who had stool specimens collected at enrollment and after therapy were included in the analysis. None had C. difficile toxin in stool specimens at enrollment. Six (27%) of 22 children with diarrhea, and 4 (7%) of 54 children without diarrhea had C. difficile toxin present at completion of therapy (P = 0.03). CONCLUSION: Toxigenic C. difficile was identified in 13% of children at the conclusion of amoxicillin/clavulanate therapy with a significantly higher frequency in children with diarrhea.

Amoxicillin↗
PubMed1991

In vitro and in vivo evaluation of tiacumicins B and C against Clostridium difficile.

Tiacumicins B and C are members of a novel group of 18-membered macrolide antibiotics with in vitro activity against Clostridium difficile. The MICs against 15 strains of C. difficile were 0.12 to 0.25 microgram/ml for tiacumicin B, 0.25 to 1 microgram/ml for tiacumicin C, and 0.5 to 1 microgram/ml for vancomycin. The resistance frequency for both compounds against C. difficile was less than 2.8 x 10(-8) at four and eight times the MIC. The in vivo activities of the tiacumicins against two strains of C. difficile were compared with that of vancomycin in a hamster model of antibiotic-associated colitis. Oral therapy with 0.2, 1, or 5 mg of tiacumicin B or C per kg of body weight protected 100% of clindamycin-treated hamsters exposed to C. difficile ATCC 9689. Oral treatment with identical doses of vancomycin produced a prolonged, dose-dependent survival of hamsters, but it did not prevent the development of fatal colitis at doses of up to 5 mg/kg. When clindamycin-treated animals were exposed to another strain of C. difficile, both tiacumicin B and vancomycin were protective at 5 mg/kg, but not at lower doses. Tiacumicin C was not tested in vivo against the second strain of C. difficile. No tiacumicin B or C was detected in the sera of hamsters treated with single oral doses of 25 mg/kg, while antibiotic levels in the ceca of these hamsters reached 248 micrograms/ml and 285 mg/ml for tiacumicins B and C, respectively. The tiacumicins demonstrated in vitro and in vivo potencies against C. difficile and achieved high concentrations in the cecum, but not the serum, of hamsters after oral administration.

Aminoglycosides↗
PubMed1994

Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection.

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum- and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.

Adolescent↗
PubMed2005

Monocytes are highly sensitive to clostridium difficile toxin A-induced apoptotic and nonapoptotic cell death.

In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 microg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.

Apoptosis↗
PubMed2000

[Joint manifestations related to Clostridium difficile].

BACKGROUND: Reactional joint disease subsequent to digestive tract infections have been well studied for Salmonella, Shigella, Yersinia and Campylobacter. Association with HLA B27 is well documented. A review of the literature since 1976 disclosed 23 cases of articular and extra-articular inflammatory reactions following pseudomembranous Clostridium difficile infection. CASE REPORT: We describe 2 new cases which followed a favorable course. This is the usual outcome although inflammatory manifestations may persist or relapse. DISCUSSION: The pathophysiological interest of this condition lies in the pathogenic potential of Clostridium difficile which appears to be directly related to toxin production. This would suggest an immune mechanism rather than pure infection.

Adult↗
PubMed2000

[Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens].

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.

Anti-Bacterial Agents↗
PubMed1991

New Clostridium difficile serotypes in Poland.

Polish isolates of Clostridium difficile strains of different origin were studied. All strains were serotyped according to the Delmee scheme. Twenty-one strains remained untypeable. By typing them with antisera against 5 Polish strains we observed 5 new serotypes.

Adolescent↗
PubMed1998

Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities.

Toxins A and B of Clostridium difficile are UDP-glucose glucosyltransferases that exert their cellular toxicity primarily through their abilities to monoglucosylate, and thereby inactivate, Rho family small GTPases. Toxin A also hydrolyzes UDP-glucose, although this activity is not well characterized. In this study, we measured the kinetics of UDP-glucose hydrolysis by toxins A and B and found significant differences in the catalytic activities of these two structurally homologous toxins. The toxins displayed similar Michaelis constants (Km) for UDP-glucose, but the maximal velocity (Vmax) of toxin B was approximately 5-fold greater than that of toxin A. Toxins A and B exert their enzymatic actions intracellularly, and, interestingly, we found that each toxin absolutely required K+ for optimal hydrolase activity; Na+ was inactive. The toxins also required certain divalent cations for activity and exhibited a significantly greater Vmax and lower Km in the presence of Mn2+ as compared with Mg2+. We conclude that C. difficile toxins A and B are cation-dependent UDP-glucose hydrolases that differ significantly in their catalytic activities, a finding that may have important implications in understanding their different cytotoxic effects.

Amino Acid Sequence↗
PubMed2004

The emergence of Clostridium difficile as a pathogen of food animals.

Clostridium difficile causes pseudomembranous colitis in humans, usually after disruption of the bowel flora by antibiotic therapy. Factors mediating the frank disease include the dose and toxigenicity of the colonizing strain, its ability to adhere to colonic epithelium, the concurrent presence of organisms that affect multiplication and toxin production or activity, and the susceptibility of the host. Toxins A (an enterotoxin) and B (a cytotoxin) play the major role in pathogenesis and the detection of toxins in gut contents is the gold standard for diagnosis. Disease in horses takes the form of often-fatal foal hemorrhagic enteritis. Nosocomial, antibiotic-associated, disease is increasingly common in adult horses. Enteric clinical signs are reported in ostriches, companion animals and recently calves. Clostridium difficile colitis is now a common diagnosis in neonatal pigs in the USA and elsewhere. Clinical features include onset at 1-5 days of age, sometimes with dyspnea, mild abdominal distension and scrotal edema, and commonly with yellow, pasty diarrhea. There is mesocolonic edema grossly, with microscopic diffuse colitis, mucosal edema, crypt distension, epithelial necrosis and superficial mucosal erosion. Neutrophil infiltration of the lamina propria is common, and fibrin and numerous rod-shaped bacteria are observed on the surface. About two-thirds of litters and one-third of piglets will be affected (based upon positive toxin tests), although this appears to vary with the season. The case fatality rate is probably low if considering only direct effects of C. difficile infection. The significance of toxin-positive non-diarrheic pigs and the nature of the interaction of toxins A and B with enterocytes are unknown. Given the widespread occurrence of the disease, there is substantial effort to develop immunoprophylactic products.

Animals↗
PubMed1998

Reduction in vancomycin-resistant Enterococcus and Clostridium difficile infections following change to tympanic thermometers.

OBJECTIVE: To contain a nosocomial outbreak of vancomycin-resistant Enterococcus (VRE). DESIGN: Intervention study, with comparison of incidence rates before and after intervention to assess whether changes in incidence followed the intervention and were greater than expected based on trends observed before the intervention. SETTING: A 343-bed acute-care hospital serving a predominantly elderly population referred from nursing homes, as well as patients admitted from the community. METHODS: Interventions strategies were tested on three high-risk nursing stations. These included enhanced environmental sanitation; intensive staff retraining in Universal Precautions, body substance isolation, and proper use of gloves; and the use of tympanic thermometers to avoid possible rectal or oral VRE transmission during temperature taking. RESULTS: Nosocomial VRE infections were reduced by 48% 9 months after switching to tympanic thermometers; incidence of Clostridium difficile infections also was reduced. As a result, tympanic thermometers were introduced facilitywide; additional observation for 20 months showed a risk reduction of 60% for VRE and 40% for C difficile. CONCLUSION: Cross-transmission of VRE and C difficile during temperature taking may result in bowel colonization, placing the patient at increased risk for infection. This risk may be reduced by the use of tympanic thermometers.

Anti-Bacterial Agents↗
PubMed1993

Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay.

This study describes the use of a new and easy method called random amplified polymorphic DNA (RAPD) assay to distinguish strains of C. difficile. We used two single short primers (AP4 and AP5) with arbitrary nucleotide sequences in a polymerase chain reaction to amplify genomic DNA. The profiles observed after electrophoretic separation were able to distinguish 20 reference C. difficile strains previously serotyped by Delmée's method. The fingerprints of 11 epidemiologically unrelated C. difficile strains clearly yielded a DNA polymorphism between all the strains. Latterly, RAPD profiles of 11 C. difficile strains isolated from 2 independent suspected outbreaks showed, in each case, a predominant banding pattern corresponding to an epidemic strain. These results suggest that RAPD assay could be a valuable tool for epidemiological studies.

Base Sequence↗
PubMed1997

Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain.

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.

Administration, Oral↗
PubMed1991

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.

Antibody Specificity↗
PubMed2001

Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae from stools submitted for Clostridium difficile testing compared to results from a focused surveillance program.

It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for Clostridium difficile testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant Enterobacteriaceae (MDRE) with both methods. Of the stools submitted for C. difficile testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for C. difficile testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for C. difficile testing. Therefore, while screening stools submitted for C. difficile testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.

Clostridioides difficile↗
PubMed2004

Colonic IgA producing cells and macrophages are reduced in recurrent and non-recurrent Clostridium difficile associated diarrhoea.

BACKGROUND: In Clostridium difficile associated diarrhoea (CDAD), histological changes in the colonic mucosa range from minimal inflammation to pseudomembranous colitis (PMC). The disease also recurs in a considerable proportion of patients. AIM: To investigate mucosal immune system cells in colonic biopsies of patients with CDAD. METHODS: Colonic biopsies were obtained from 12 control patients with diarrhoea, six patients with CDAD and minimal inflammation, and 10 patients with CDAD with pseudomembranous colitis (samples obtained from areas with and without inflammatory exudate). Immunohistochemical studies were performed using antibodies to T cells (CD3), macrophages (CD68), B/plasma cells (CD79alpha), and to IgA, IgM, and IgG. Labelled cells in lamina propria were quantified. RESULTS: In contrast to T cells, there were significant reductions in B/plasma cell and macrophage counts in all biopsies from patients with CDAD compared with controls (p<0.001). Studies using anti-immunoglobulin antibodies showed significant reductions in IgA producing cells in CDAD biopsies (p<0.05), with the greatest reduction in samples from patients with PMC. In contrast, there was a significant increase (p<0.05) in IgG producing cells in CDAD biopsies. Only patients with PMC relapsed. In these patients, B/plasma cell and IgA producing cell counts (in biopsies with and without inflammatory exudates) were significantly lower (p<0.01) in mucosal samples from those who subsequently relapsed (five) than those who did not. CONCLUSIONS: A selective reduction in mucosal IgA producing cells and macrophages is associated with colonic disease in C difficile infected patients. Severe reduction in colonic IgA producing cells may predispose to recurrence of CDAD.

Adolescent↗